Radioiodinated monoclonal antibodies are important for the diagnosis and therapy of cancer as summarized by Goldenberg in Amer. J. Med. 1993; 94: 297-312. A number of methods have been developed over the last thirty years to chemically introduce radioiodine into monoclonal and polyclonal antibodies for these uses. Iodine is preferred as a radiolabel in these applications because the chemistry used for radioiodination of protein is relatively easy, radioiodine has useful physical decay characteristics, and isotopes of iodine are commercially available. Various chemistries have been developed to link iodine to antibodies that target cancer cells. These chemistries have been reviewed by Wilbur, Bioconjugate Chemistry 1992; 3: 433-70. The most common linking procedure has been to prepare in situ an electrophilic radioiodine species to react with a functional group on an antibody. Reagents such as chloramine-T and iodogen have been employed to generate electrophilic iodine. A tyrosine group on protein is usually the site of iodination.
Conventional radioiodinations of MAbs require the removal of oxidant used as well as unincorporated radioiodide using some purification method. When using buffer-soluble oxidant such as chloramine-T, the oxidant and radioiodide are customarily removed by size-exclusion chromatography on a size-exclusion column, such as commercially available PD10® column.
A major drawback with using the direct radioiodination schemes is the phenomenon of in vivo deiodination. As a result of antibody internalization and lysosomal processing in vivo, a labeled protein is degraded to small peptides, and its radioiodine is released from the cell in the form of iodotyrosine or as iodine attached to a low molecular weight peptide fragment. These findings have been reported by Geissler et al., Cancer Research 1992; 52: 2907-2915 and Shih et al., J. Nucl. Med. 1994; 35: 899-908. Such in vivo removal of radioiodine from target cells reduces tumor-to-nontumor discrimination which is important for radiodiagnosis, and also reduces the residence time of radioiodine in target cells which significantly affects radiotherapy effectiveness.
Several approaches have been devised to overcome the phenomenon of in vivo deiodination, through the design of iodine radiolabels which are intracellularly retained. Such labels are referred to as “residualizing labels”. In one method, radioiodine is attached to non-metabolizable carbohydrates, and the latter are first activated and then conjugated to antibodies. This approach is exemplified by lactitoltyramine (LT) and dilactitoltyramine (Strobel et al., Arch. Biochem. Biophys 1985; 240: 635-45) and tyrosine cellobiose (Ali et al., Cancer Research 1990; 50: 783s-88s). In another approach, a pyridine-based moiety, “SIPC”, was utilized (Reist et al., Cancer Research 1996; 56:4970-4977). Pentapeptides containing all D amino acids and multiple basic amino acids, have also been explored (Foulon et al., Cancer Research 2000; 60:4453-4460). In yet another approach, DTPA-appended, radioiodinated peptides containing D-amino acids were successfully utilized as residualizing labels (Govindan et al., Bioconjugate Chemistry 1999; 10:231-240; Stein et al., Cancer Research 2003; 63:111-118).
When a radioiodinated small molecular mass material is conjugated to MAbs (hereinafter radioiodinated conjugates), as illustrated in the references given in the previous paragraph, an additional requirement presents itself in that the unconjugated material needs to be removed as well. This invariably requires an additional column method of purification. The carbohydrate method results in low overall yield and specific activity, and involves a column method of purification at the end of the process. The methods of Reist et al (supra) and Foulon et al (supra) involve two column purification steps, one at the radioiodination stage and the other at the antibody conjugation stage. The method of Govindan et al. (supra; further described in Stein et al. (supra)) involves one column purification at the end of the process, with higher overall yields and specific activities.
Column methods are generally cumbersome, and have additional drawbacks of radiation exposure to personnel, especially when handling hundreds of mCi of I-131 for clinical-scale preparations, and catastrophic column failures. In iodogen-based radioiodinations, the oxidant is water-insolube, and is removed by simply syringing out or filtering off the radiolabeled material. In these instances, the only other material that needs to be removed is unincorporated radioiodide. Because of the relatively higher affinity of iodide versus phosphate or hydroxide ion to bind to strong anion-exchange resin, unincorporated iodide has been shown to be removable by using an anion-exchange resin [Weadock et al. J Nuclear Medicine 1990; 31:508-511); Behr™ et al. Nuklearmedizin 2002; 41:71-79). It is also conceivable to use immobilized chloramine-T oxidant such as commercially available “IODO-BEADS”® in combination with anion exchange resin. While a certain simplification is achieved in the purification of directly radioiodinated antibodies by such combinations, the use of radioiodinated conjugates still requires the removal of unconjugated small molecular mass moieties. In as much as this purification is usually achieved by column methods, the attendant drawbacks of the methods pose practical problems in the purification of several hundreds of millicuries of radiolabeled preparations for clinical applications.
A column method has been described by Li et al. (Bioconjugate Chemistry 1994; 5: 101-104) to purify a radiometal-chelated DOTA-peptide from unlabeled DOTA-peptide by passing through a column of diethylaminoethyl-cellulose anion-exchanger, and eluting with several portions of water. In this case, radiometal-chelated DOTA peptide has a neutral charge, and is therefore eluted from the column, while unlabeled material, with negative charge on the chelator portion, is retained by the anion-exchange column. This was necessitated by the need to purify radiometal-chelated bifunctional material, which was subsequently conjugated to antibodies, and the product was purified by size-exclusion column method. Thus, the procedure of Li et al. (supra) involves a multi-step approach and two column-based purification steps. Again, such column-based methods will be impractical when applied to large-scale radioiodination of small molecular mass moieties followed by conjugation to targeting agents. In the latter case, involving hundreds of millicuries of radioactive iodine, simpler purification methods are necessitated.